Method for producing phospholipid

ABSTRACT

A method for producing a phospholipid using transphosphatidylation, which comprises homogenizing a mixture of a raw material phospholipid, a hydroxyl-containing acceptor, phospholipase D, and water in the absence of an organic solvent to obtain a homogenized mixture; and subjecting the homogenized mixture to a transphosphatidylation reaction at 15° C. to 65° C. The homogenized mixture has a lamellar lyotropic liquid crystal structure. An objective phospholipid can be obtained from the homogenized mixture through transphophatidylation without using an organic solvent or calcium.

CROSS-REFERENCE TO RELATED APPLICATIONS

This is a continuation application of application Ser. No. 10/344,063filed Feb. 6, 2003, which is a U.S. national phase application under 35U.S.C. 371 of International application No. PCT/JP01/06502 (notpublished in English) filed Jul. 27, 2001.

TECHNICAL FIELD

The present invention relates to a method for producing an objectivephospholipid using transphosphatidylation (phospholipid base exchangereactions).

BACKGROUND ART

Phospholipids such as phosphatidylserines (PSs) andphosphatidylglycerols (PGs) each have their useful physiological orbiological functions and specific physical properties and are used in,for example, pharmaceutical preparations, food materials, andemulsifying agents. For example, phosphatidylserines are promising asdrugs for prophylaxis and/or therapy of senile dementia and dysmnesia(memory disorder), phosphatidylglycerols are promising as emulsifyingagents, and phosphatidylascorbic acids are promising as emulsifyingagents and lipoperoxides inhibitors.

These phospholipids have been conventionally produced by chemicalsynthesis or by transphosphatidylation using phospholipase D. Amongthese production methods, enzymatic methods can relatively easilyproduce phospholipids at relatively low cost and are widely used.

Methods for producing objective phospholipids by transphosphatidylation(phospholipid base exchange reactions) have been known from a long timeago (Yang, S. F. et al., J. Biol. Chem., 242, p.477, 1967). For example,Kokusho et al. disclose that a reaction product containingphosphatidylserine is obtained by a biphasic reaction in whichphospholipase D is allowed to act upon a mixture of a solution ofegg-yolk phosphatidylcholine in isopropyl ether with a L-serine aqueoussolution containing calcium chloride (Agric. Biol. Chem., 51, p.2515,1987). It is generally believed that a reaction system in such abiphasic reaction comprises two phases of an oil phase containing amaterial phospholipid and an aqueous phase containing an acceptor andthat transphosphatidylation occurs at the interface between the twophases.

Japanese Patent No. 2,942,302 describes a homogenous reaction in which aphospholipid preparation containing about 85% of phosphatidylcholineprepared by fractionating soybean lecithin is dissolved in ethylacetate, the resulting solution is mixed with an ascorbic acid aqueoussolution to yield a mixture, and the mixture is allowed to react withphospholipase D to thereby yield a reaction product containingphosphatidyl ascorbate.

However, the biphasic reaction must be carried out in the presence ofsolvents (an organic solvent and water) five times or more(volume/weight) as much as the phospholipid and thereby must use areactor having a volume capacity six times as much as the amount of thephospholipid. In addition, calcium added to accelerate the reactionrapidly forms a salt with the phospholipid. The formed calcium salt isbelonging to the category of chemically synthesized substances in Japanand Europe, and the product is thereby difficult to use in food.

In the homogenous reaction (monophasic reaction), the reaction systemcontains large amounts of water and yields a phosphatidic acid, as aby-product, due to hydrolytic activity of phospholipase D during acontinuous reaction, thus the separation and purification of theobjective phospholipid becoming difficult. In addition, the proportionof the acceptor to the phospholipids is limited in the homogenousreaction, and thereby the production amount of the objective reactionproduct (phospholipid) is limited.

A patent granted to Fujita et al. (JP-B-7-016426) describes thatphosphatidylserine, phosphatidylglycerol, and others are produced by areverse micelle reaction in which an aqueous phase containing calciumchloride, a hydroxyl-containing acceptor, and phospholipase D and beingencapsulated in a reversed micelle is allowed to react with a solutionof a raw material phospholipid in an organic solvent (diisopropyl ether,isooctane, cyclohexane, benzene, chloroform-isooctane, n-hexane, ordichloromethane-isooctane).

In the Japanese Patent Publication, Fujita et al. report that thereverse micelle reaction requires only a small amount of water andthereby suppresses the formation of phosphatidic acids, the problem ofthe above method. However, the method in question insufficiently yieldsthe objective phospholipid in a yield of at most about 20%, requirescomplicated operations such as ultrasonic treatment and thus invitesproblems in operability and cost. The method also requires the organicsolvent 10 times (volume/weight) as much as the phospholipid and mustuse a reactor having a capacity many times as much as the amount of thephospholipid.

These conventional transphosphatidylation reactions must be carried outin a reaction system containing an organic solvent. However, whenproduct phospholipids are used in, for example, food and pharmaceuticalpreparations, the organic solvent must not remain in the products andmust be completely removed. Accordingly, their production process stepsrequire facilities for the removal of the organic solvent, thus invitingdisadvantages in, for example, operability and cost. In particular, suchorganic solvents cannot be substantially used in the reactions based onthe food sanitation law when the products are used in the production offood.

Demands have therefore been made on methods for producing phospholipidswithout using organic solvents and/or calcium salts. However, oneskilled in the art generally believes that a reaction does not smoothlyproceed without using organic solvents and thereby the yield of theobjective phospholipid and operability should decrease, since thematerial phospholipids such as phosphatidylcholines are oil-soluble.

DISCLOSURE OF INVENTION

Accordingly, an object of the present invention is to provide a methodfor producing phospholipid by transphosphatidylation, usingphospholipase D, which can easily produce the objective phospholipid ina high yield without using organic solvents and/or calcium.

Thus, according to the present invention, the above object can beachieved by a method for producing phospholipid throughtransphosphatidylation, comprising the steps of homogenizing a mixtureof a raw material phospholipid, a hydroxyl-containing acceptor (accepterhaving one or more hydroxyl groups), phospholipase D, and water in theabsence of an organic solvent to obtain a homogenized mixture; andsubjecting the homogenized mixture to a transphosphatidylation reactionat a temperature within a range from 15° C. to 65° C.

In the method according to the present invention, the four components,the raw material phospholipid, the hydroxyl-containing acceptor,phospholipase D, and water, are sufficiently mixed, and the resultingmixture is further homogenized. The homogenized mixture is supposed tohave a lamellar lyotropic liquid crystal structure. The term “lamellarlyotropic liquid crystal” means a liquid crystal of a phospholipidbilayer membrane formed by adding water to a phospholipid. In thepresent invention, it is supposed that the homogenized mixture has anarray structure containing the bilayer membrane (sometime apolymolecular layer membrane structure) and an aqueous layer alternatelycontinuously arrayed. The lamellar lyotropic liquid crystal structurecan be identified by, for example, microscopic observation of thehomogenized mixture under crossed Nicols.

In this connection, a “lamellar lyotropic liquid crystal structuresubstantially without phase separation” mentioned later and a “lamellarlyotropic liquid crystal structure with phase separation” are observedas a continuous lamellar structure and as a closed-ring structurefloated in an aqueous phase, respectively.

The lamellar lyotropic liquid crystal structure is generated in thefollowing manner. That is, by adding water to the raw materialphospholipid in the homogenization procedure, the interaction betweenhydrophilic groups in the phospholipid becomes weak, and its crystalstructure disintegrates to forme a lamellar liquid crystal. Because ofthe formation of the lamellar lyotropic liquid crystal structure, watercan freely move between layers of the lamellar structure of thephospholipid to make it efficient to supply the acceptor and/or theenzyme and to remove polar heads liberated from the phospholipid,thereby enabling a transphosphatidylation reaction.

To increase the yield of the reaction product, the entire homogenizedmixture must have the lamellar lyotropic liquid crystal structure. Forthat purpose, the individual components must be homogenized in additionto simply mixed. In other words, the entire homogenized mixturepreferably has the lamellar lyotropic liquid crystal structure (i.e.,the mixture substantially has the lamellar lyotropic liquid crystalstructure) as a result of homogenization for a higher yield of thereaction product.

A water content in the homogenized mixture affects the formation of thelamellar lyotropic liquid crystal structure. If the water content iswithin a specific range (e.g., from about 10 wt % to about 100 wt %relative to the amount of soybean phospholipid), the homogenized mixturewill have a lamellar (neat) liquid crystal with substantially no phaseseparation. However, if the water content is excessively higher than thespecific range, the homogenized mixture may undergo phase separation toform two phases containing a liquid and a liquid crystal. In the methodaccording to the present invention, the reaction is preferably performedwhile the homogenized mixture is without phase separation or withsubstantially no phase separation (hereinafter both are referred to as“lamellar lyotropic liquid crystal structure substantially without phaseseparation”) for a further higher transfer activity.

In contrast, in a homogenized mixture containing an excessively largeamount of water to thereby invite phase separation (a lamellar lyotropicliquid crystal structure with phase separation), the liquid crystal mayconstitute discontinuous small granules floating in the solvent.Accordingly, the contact efficiency between water and the phospholipiddecreases, thus decreasing transfer activity as compared with the casewithout phase separation. If the water content is excessively low, thephospholipid may maintain its crystal structure to inhibit the formationof a lamellar lyotropic liquid crystal structure overall thephospholipid to thereby lose the field of an enzymatic reaction.Alternatively, the homogenized mixture may have decreased fluidity andthereby have a deteriorated contact efficiency between the substrate andthe enzyme, thus the enzyme cannot efficiently act upon the substrate.

Organic solvents are preferably not used in the homogenizationprocedure. If an organic solvent is added during the homogenizationprocedure, phase separation as in conventional biphasic reactions (notphase separation in the lamellar lyotropic liquid crystal structure butseparation between an oil phase and an aqueous phase) may be enhanced.In addition, the addition of an organic solvent invites variousdisadvantages as described above.

Raw material phospholipids for use in the present invention include anyof phospholipid-containing natural products, extracts or purifiedextracts of such natural products, and synthetic phospholipids. Examplesof the phospholipids include, but are not limited to, soybean lecithin,rapeseed lecithin, egg yolk lecithin, corn lecithin, cottonseedlecithin, purified products of these lecithins; phosphatidylcholines(hereinafter briefly referred to “PC(s)”), phosphatidylethanolamines(hereinafter briefly referred to as “PE(s)”), and mixtures of thesesubstances. Among them, soybean lecithin, rapeseed lecithin, egg yolklecithin, and purified products of these lecithins are preferred fortheir availability and cost

Hydroxyl-containing acceptors for use in the present invention are notspecifically limited as long as they can receive or accept aphosphatidyl group of the raw material phospholipid in the presence ofphospholipase D. Such hydroxyl-containing acceptors include, forexample, serine, glycerol, L-ascorbic acid, glucose, choline,ethanolamine, 1-amino-2-propanol, and 1-o-methyl-glycoside. Among them,serine, choline, L-ascorbic acid, glucose, and glycerol are preferredfor a higher yield of the objective product, of which serine andglycerol are typically preferred.

Phospholipase Ds (hereinafter briefly referred to as “PLD(s)”) for useherein are not specifically limited as long as they haveransphosphatidylation activity and include, for example, free orchemically modified PLDs, and immobilized enzymes immobilized to acarrier such as ion exchange resins and silica gel. Among them, freePLDs are preferred for a further higher yield of the reaction product.

More specifically, any of PLDs derived from plants or vegetables such ascabbage and carrot, PLDs derived from microorganisms such asmycobacteria, bacteria, yeasts, and fungi (molds), and PLDs derived fromanimals can be advantageously used. They may be prepared productsprepared according to a conventional procedure or commercially availableproducts such as a cabbage-derived PLD (e.g., Product Number P 7758,Sigma Chemical Company), peanut-derived PLD (e.g., Product Number P0515, Sigma Chemical Company), and a PLD derived from Streptmyceschromofuscus.

According to the present invention, the raw material phospholipid, thehydroxyl-containing acceptor, PLD, and water are initially mixed, andthe resulting mixture is further subjected to a homogenizationprocedure. The homogenization procedure herein means homogenousdispersion of the four components in the mixture by the application ofphysical force by means of, for example, physical agitation, ultrasonictreatment, etc. More specifically, homogenization treatments using aVibromixer, automatic mortar, Homo Mixer, Physcotron, food processor, orsonicator alone or in combination can be employed. If the amount of themixture is small, it may be kneaded using, for example, a microspatula.The four components may be mixed and homogenized separately in time orsimultaneously. For example, one of the four components may be mixed andhomogenized with another, and this procedure is repeated. It is alsoacceptable that two, three or four of the components are mixed ordissolved in advance, and then the resulting mixture or solution isultimately homogenized.

If the homogenization procedure is not performed in the method of thepresent invention, the yield of the objective phospholipid significantlydecreases. This is probably for the following reasons. That is, the rawmaterial phospholipid is generally a hard paste at room temperature, andthe entire mixture cannot have a lamellar lyotropic liquid crystalstructure by simply mixing the other components. As a result, theindividual components come in contact with one another at a lowerfrequency to thereby decrease the yield of the objective product.Accordingly, the mixture of the components must be applied with physicalforce to such an extent as to disperse the individual components andmust be subjected to a “homogenization procedure”.

The hydroxyl-containing acceptor can be used as an aqueous solution oras a powder in the homogenization procedure. PLD is used as a solutionin a small amount of water or as a powder. In this procedure, if theother three (the raw material phospholipid, PLD, and water) than theacceptor have been mixed in advance, phosphatidic acid (hereinafterreferred to as “PA”) is by-produced. Accordingly, it is preferred thatthe raw material phospholipid is mixed with the acceptor in advance, andthe resulting mixture is then mixed with the other components, or thefour components are mixed and homogenized simultaneously.

As is described above, the amount of water content in the homogenizationprocedure affects the phase separation of the lamellar lyotropic liquidcrystal structure and the yield of the reaction product phospholipid andis preferably controlled and adjusted to prevent the phase separation.While depending on the type of the raw material phospholipid, the watercontent in the lamellar lyotropic liquid crystal structure is preferablyfrom 10 wt % to 100 wt % and more preferably from 20 wt % to 60 wt %relative to the amount of the raw material phospholipid to substantiallyprevent the phase separation. The term “water content” in the lamellarlyotropic liquid crystal structure as used herein means the amount ofwater in the homogenized mixture after removing water separated duringthe homogenization procedure and can be determined by, for example, aconventional Karl Fischer technique. The separated water may be removedby decantation or centrifugal separation, for example, at 150 g forabout 1 minute.

It is not necessarily appropriate to specify a preferred amount of theacceptor, since it varies depending on the type of the acceptor. For abetter yield of the reaction product and operability, the amount ispreferably from about 0.3 mole to about 10 moles and more preferablyfrom about 4 moles to about 8 moles per 1 mole of the raw materialphospholipid. If an excessively large amount (exceeding 10 moles) of theacceptor is added, the recovery of an unreacted acceptor may requireincreased efforts. In contrast, if the amount is excessively small, theyield of the objective product may decrease.

More specifically, the amount of serine as the hydroxyl-containingacceptor is preferably from 5 wt % to 150 wt %, and more preferably from50 wt % to 100 wt % relative to the amount of the raw materialphospholipid. The amount of glycerol as the hydroxyl-containing acceptoris preferably from 10 wt % to 200 wt %, and more preferably from 20 wt %to 100 wt %.

The amount of PLD is not specifically limited, can be determineddepending on, for example, the reaction time in the subsequent reactionstep and is generally from about 500 to 100,000 units per kg of thephospholipid.

A temperature in the homogenization procedure is not specificallylimited and is preferably from about 15° C. to 65° C. If the temperatureis lower than 15° C., it may require extra energy to cool the reactionsystem, and the mixture may not sufficiently be homogenized. If it ishigher than 65° C., the phospholipid may become unstable.

In addition to the aforementioned components, an edible oil and/or fatcan be added during the homogenization procedure within ranges notdeteriorating the lamellar lyotropic liquid crystal structure. Althoughthe addition of such an edible oil and/or fat enhances the conversion ofthe lamellar lyotropic liquid crystal structure into a biphasic systemas organic solvents, a small amount of the edible oil and/or fatincreases the fluidity of the homogenized mixture to thereby increasethe yield of the objective phospholipid. Such edible oils and fatsinclude, but are not limited to, safflower oil, soybean oil, corn oil,rapeseed oil, cottonseed oil, sunflower oil, safflower oil, sesame oil,olive oil, hempseed oil, perilla oil, theobroma oil (cacao butter),coconut oil, and other vegetable oils; butter oil, fish oil, lard, beeftallow, and other animal oils; and middle-chain triacylglycerols (MCTs).Each of these oils and fats is added alone or in combination to the rawmaterial phospholipid to impart fluidity thereof. Among them, MCTs,theobroma oil, and soybean oil are preferred to further improve theyield of the objective product

While depending on the types of the raw material phospholipid and theadded edible oil and/or fat, the amount of the edible oil and/or fat ispreferably less than or equal to an equivalent amount and morepreferably from 5 wt % to 15 wt % relative to the amount of the rawmaterial phospholipid.

It is possible to add an organic solvent such as hexane or ether for thesame purpose within ranges not deteriorating the lamellar lyotropicliquid crystal structure, but is not preferred as described above.

Some of advantages of the present invention will be describedhereinafter. Conventional transphosphatidylation reactions have beenperformed according to any of biphasic reactions, reversed micellereactions, and homogenous phase reactions. The biphasic reactions andreversed micelle reactions require a solvent five times (volume/weight)or more as much as the phospholipid to maintain their reaction systems.In contrast, the method of the present invention requires a solvent onlyone time as much as the phospholipid and can thereby use a reactorhaving a smaller capacity for the production of the phospholipid in thesame amount as in the conventional equivalents.

The homogenous phase reactions require a solvent two times(volume/weight) or more as much as the phospholipid and thereby requirea reactor having a capacity three times (volume/weight) or more as muchas the phospholipid. In addition, the amount of the acceptor is limitedto maintain their homogenous phase, and it is difficult to obtained areaction product containing the objective compound in a high content.For example, to produce PS, the amount of serine that can be added to 1kg of the phospholipid is 0.15 kg and the PS content in the reactionproducts phospholipids is at most 35%. In contrast, according to themethod of the present invention, 1 kg or more of serine can be added to1 kg of the phospholipid, and thereby reaction products containing 48%or more of PS in the phospholipids can be obtained. Specifically, themethod of the present invention can reduce the capacity of a reactor toproduce an equivalent amount of the objective phospholipid and can yieldreaction products containing the objective compound in a high content ascompared with the homogenous reactions.

Each of the conventional transphosphatidylation reactions must beperformed in the presence of an organic solvent such as an ether,toluene, n-hexane, or ethyl acetate in their reaction system. However,the method according to the present invention does not require organicsolvents and is therefore advantageous in the production of thephospholipids for food and other substances in which the use of organicsolvents is restricted. Further, according to the present invention, thereaction proceeds even without the addition of calcium ions in contrastto the conventional methods. While not being clarified, this is probablybecause the reaction is performed in a liquid crystal according to thepresent invention and thereby the raw material phospholipid becomesstructurally susceptible to transphosphatidylation reactions regardlessof the presence or absence of calcium ion. In contrast, the conventionalbiphasic reaction is performed in a liquid.

According to the present invention, the homogenized mixture thusobtained is subjected to a transphosphatidylation reaction in theabsence of an organic solvent to yield the objective phospholipid. Thereaction is performed at a temperature preferably within a range from15° C. to 65° C. and more preferably from 45° C. to 55° C. If thereaction temperature is lower than 15° C., the reaction may not beenhanced to proceed to thereby deteriorate the yield of the objectivephospholipid. If the reaction temperature is higher than 65° C., sidereactions of phospholipid, such as decomposition and/or oxidation of theproduced phosphatidylserine, may occur.

A reaction time is not specifically limited, can be appropriatelyselected depending on the reaction temperature, the types and amounts ofthe components and is preferably from about 1 to 48 hours for a betteryield of the objective phospholipid and less by-production of PA.

The transphosphatidylation reaction can be performed left stand or withstirring. For example, the reaction can be performed with stirring usinga tabletop universal mixer (e.g., KINMIX MAJOR (trademark),Type-KM-230).

In the production of phospholipids according to the method of thepresent invention, a lamellar liquid crystal structure without phaseseparation is formed entirely in the homogenized mixture by mixing anappropriate amount of water with the raw material phospholipid, andwater can freely move between layers of the lamellar structure of thephospholipid, thereby efficiently supplying the hydroxyl-containingacceptor and/or the enzyme as well as efficiently removing polar headsliberated from the phospholipid. Accordingly, it is possible to achievethe transphosphatidylation reaction at high activity, and the objectivephospholipid can be easily obtained in high yield at low cost.

Phospholipids obtained according to the present invention may containimpurities derived from the raw material phospholipid and the acceptor,and other substances in addition to by-product PA. The obtainedphospholipid is therefore often a phospholipid mixture containing theobjective phospholipid, other phospholipids and impurities.

The phospholipid obtained by the transphosphatidylation is preferablyused after the removal of such impurities by subjecting the same to anappropriate purification process but can be used while containingimpurities derived from the raw materials or formed in the productionprocesses as long as the impurities do not invite problems inadministration or deteriorate the advantages. The product can bepurified by any technique such as fractionation using a solvent andchromatography in appropriate combination according to a conventionalprocedure.

The phospholipids obtained according to the present invention can beadministered in the form of, for example, pharmaceutical preparations,food, or cosmetics. For example, to be used in the form ofpharmaceutical preparations to utilize the biological functions of thephospholipids, they can be orally administered in the dosage form of,for example, capsules, granules, tablets, powders, and other solidpreparations; and syrups, and other liquid preparations. Alternatively,they can be administered in the form of, for example, injections,dermatologic external preparations, rectal infusions, and other non-oralpreparations.

In production of these preparations, additional pharmaceuticallyacceptable components can be used according to necessity. Suchcomponents include, for example, lactose, starches, crystallinecellulose, calcium lactate, magnesium aluminometasilicate, silicicanhydride, and other excipients; sucrose, hydroxypropylcellulose,polyvinylpyrrolidone, and other binders; carboxymethylcellulose,carboxymethylcellulose calcium, and other disintegrators; magnesiumstearate, talc, and other lubricants.

When the phospholipid obtained according to the method of the presentinvention is used in food in the expectation of similar biologicalfunctions, such food can be appropriately produced according to aconventional procedure by adding the phospholipid as intact or afterpurification to oils and fats, tablet or granular confectionery,fermented milk, candies, spices, fish and vegetables flakes to sprinkleon cooked rice, and other food and drinks.

An appropriate amount of the phospholipids obtained by the method of thepresent invention can be used in the form of these pharmaceuticalpreparations and food. To obtain the biological functions of thephospholipids, the amount thereof may be such as to yield the functionsand not to invite problems such as overdose. For example, thecompositional amount of phosphatidylserine produced as the phospholipidis such an amount as to take in from about 50 mg to 1,000 mg a day.

The phospholipids obtained by the method according to the presentinvention can also be used as emulsifying agents. In this case, theemulsifying agent may be added to, for example, pharmaceuticalpreparations, food, and cosmetics in an amount preferably from 0.01% to10%.

Among phospholipids obtained by the method according to the presentinvention, a phosphatidylserine can be easily purified and concentratedfrom a phospholipid mixture containing the phosphatidylserines bydissolving the phospholipid mixture in an alcohol to yield a solution,adding a metallic salt to the solution to insolubilize thephosphatidylserines, and separating the insolubilized matter.

Such metallic salts for use herein include, but are not limited to,lithium salts, sodium salts, potassium salts, calcium salts, magnesiumsalts, and other metallic salts, as well as natural products containingany of these metallic salts in abundance, such as common salt, bittem,brine, dolomite, and edible powdery mother of pearl. Among them, lithiumsalts, sodium salts, and potassium salts are preferred for efficientconcentration, of which lithium chloride, sodium chloride, and potassiumchloride are typically preferred. Each of these metallic salts can beused alone or in combination.

The amount of the metallic salt(s) is not specifically limited as longas phosphatidylserine can be precipitated and is preferably from 0.15 to10 mmol, and more preferably from 0.5 to 5 mmol per gram of thephospholipid for a higher recovery rate of the phosphatidylserines and ahigher content of the phosphatidylserines in the precipitate.

Any alcohol can be used herein as long as it can dissolve thephospholipid mixture, of which methyl alcohol, ethyl alcohol, butylalcohol, propyl alcohol, isopropyl alcohol, and other lower alcohols arepreferred. Mixtures of these alcohols can also be used. Ethyl alcoholcan be easily applied to food with less problems in safety and istypically advantageously used.

The concentration of the phospholipid mixture in the alcohol is notspecifically limited, is preferably equal to or more than such aconcentration that the mixture can be completely dissolved in thealcohol and is preferably from 1 to 50%, and more preferably from 2 to20% relative to the weight of the alcohol for efficient concentrationprocedure of the phosphatidylserines and for higher operability.

The phosphatidylserines can be concentrated from the phospholipidmixture, for example, in the following manner. Initially, thephospholipid mixture prepared by transphosphatidylation and containingother phospholipids in addition to the phosphatidylserines is dissolvedin an alcohol such as ethyl alcohol. In this procedure, dissolutionconditions such as dissolution temperature are not specifically limitedand can be appropriately selected depending on the types and amounts ofcomponents of the mixture and other parameters.

In the resulting solution, the phosphatidylserines, PC, PA, and otherphospholipids are extracted into a solvent layer, but some insolublematters may form. Accordingly, the metallic salt is added after removingsuch precipitates, aggregates, and other insoluble matters from thesolvent by means of, for example, centrifugal separation and/orfiltration. If the insoluble matters include a small amount ofphosphatidylserine, extraction operation with the alcohol can berepeated several times.

Next, the metal salt is added to the alcohol solution to therebyfractionate the phosphatidylserines extracted in the solvent layer.Specifically, most of the other phospholipids than thephosphatidylserines in the solvent layer do not precipitate by theaddition of the metal salt, but most of the phosphatidylserinesprecipitates, and thus the phosphatidylserines can be concentrated byrecovering the precipitated phosphatidylserine. The metal salt can beadded as a powder or a solution in a solvent such as water or analcohol. Conditions in the procedure are not specifically limited andcan be appropriately selected depending on, for example, the types andamounts of components of the mixture. More specifically, the mixturewith the metallic salt is held at 10° C. to 30° C. for 30 minutes orlonger to thereby insolubilize PS.

The phosphatidylserines insolubilized as a result of addition of themetallic salt can be recovered by a means such as centrifugalseparation, filtration, and standing separation. The phosphatidylserinescan be further purified by a conventional purification means such ascolumn chromatography. The phosphatidylserines concentrate according tothe present invention contains significantly reduced amounts of otherphospholipids and other components and can thereby be relatively easilypurified.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a diagram of test results on optimum amounts of MCT with theordinate A showing the PS production rate (%) and the abscissa B showingthe added amount ratio (%) of MCT to the phospholipids.

FIG. 2 is a diagram of test results on optimum amounts of MCT with theordinate C showing the ratio (%) of MCT to the phospholipids and theabscissa D showing the PS production rate (%).

FIG. 3 is a diagram of test results on molar ratios of serine tophospholipids in a homogenized mixture with the ordinate E showing thePS production rate (%) and the abscissa F showing the molar ratio(mol/mol) of serine to the phospholipids.

FIG. 4 is a diagram of test results on weight ratios of water tophospholipids in a homogenized mixture with the ordinate G showing thePS production rate (%) and the abscissa H showing the ratio (% byweight) of water to the phospholipids.

FIG. 5 is a diagram of test results on weight ratios of water tophospholipids in a homogenized mixture with the ordinate I showing thePS production rate (%) and the abscissa J showing the ratio (% byweight) of water to the phospholipids.

FIG. 6 is a diagram of test results on weight ratios of water tophospholipids in a homogenized mixture with the ordinate K showing thePS production rate (%) and the abscissa L showing the ratio (% byweight) of water to the phospholipids.

FIG. 7 is a diagram showing production rates of PS at different reactiontemperatures with the ordinate M showing the PS production rate (%) andthe abscissa N showing the temperature (°C.).

BEST MODE FOR CARRYING OUT THE INVENTION

The present invention will now be explained in detail with reference topreferable embodiments below, which are not intended to limit the scopeof the invention.

EXAMPLE 1 Basics

A total amount of 97 g of 6.7 M L-serine aqueous solution (with orwithout 0.18 M calcium chloride) was kneaded (homogenized) with 100 g ofa mixture of soybean lecithin and cocoa butter (NATHIN 250, trademark,available from Central Soya Company, Inc.) in an automatic mortar (ModelANM-150) and was held at 55° C. A total of amount 2.0 mL of an enzymemixture containing 800 units of phospholipase D-Y1 derived from anactinomycete belonging to Streptomyces (PLD-Y1, product name, availablefrom Yakult Honsha Co., Ltd.) was kneaded (homogenized) with the kneadedmixture in an automatic mortar, and a reaction was performed in theabsence of organic solvents. The transphosphatidylation reaction wasperformed with stirring at 55° C. for 17 hours and was complete.

Analyses of reaction products by silica gel thin-layer chromatographyrevealed that 47.9% by mole of total phospholipids was converted intophosphatidylserines when 0.18 M calcium chloride was added, and that44.5% by mole of the total phospholipids was converted intophosphatidylserines even in the absence of calcium chloride. Theseresults show that the transphosphatidylation reaction can proceed inthis reaction system even without the addition of calcium. Thehomogenized mixture before the initiation of the reaction was observedwith a microscope under crossed Nicols and was found to have a “lamellarlyotropic liquid crystal structure substantially without phaseseparation”.

As is described above, the present invention provides a method forperforming a transphosphatidylation reaction without the use of organicsolvents. Specifically, transphosphatidylated products can beefficiently produced by homogenizing an aqueous solution containing thehydroxyl-containing acceptor and phospholipase D with the phospholipidbefore reaction.

The method does not require any organic solvent. However, an oil-solublesubstance other than organic solvents can be added to the phospholipidto thereby further improve the operability. Such oil-soluble substancesinclude, but are not limited to, safflower oil, soybean oil, corn oil,rapeseed oil, cottonseed oil, sunflower oil, safflower oil, sesame oil,olive oil, hempseed oil, perilla oil, theobroma oil (cacao butter),coconut oil, and other vegetable oils; butter oil, fish oil, lard, beeftallow, and other animal oils; MCTs as glyceride derivatives preparedfrom middle-chain fatty acids as starting materials; and other edibleoils and fats.

EXAMPLE 2 Addition of Calcium

The results of Example 1 show that the transphosphatidylation reactionproceeds even without the addition of calcium. However, the effects ofthe addition of calcium were further investigated. Specifically,individual components were mixed with or without the addition of calciumas shown in Table 1 while the molar ratio of serine to the phospholipidwas set at 1 or 5. The resulting mixture was kneaded (homogenized) usinga microspatula and was allowed to react at 55° C. for 18 hours. In thisprocedure, a sample with the molar ratio of 1 employed 2.5 M serineaqueous solution, and one with the molar ratio of 5 employed 4.5 Mserine aqueous solution. More specifically, NATHIN 250 and the serineaqueous solution containing, if any, calcium were mixed, and theresulting mixture was treated with the PLD-Y1 aqueous solution toinitiate a reaction.

The amounts of reaction products were determined in the same manner asin Example 1. Table 2 shows that, the production rate of PS without theaddition of calcium was slightly lower than but substantially the sameas that with the addition of calcium. Homogenized mixtures beforereaction were subjected to microscopic observation under crossed Nicolsand were found to have a lamellar lyotropic liquid crystal structuresubstantially without phase separation. In the tables, the abbreviationsPA, PE, and PI represent phosphatidic acid, phosphatidylethanolamine,and phosphatidylinositol, respectively. TABLE 1 Molar ratio of serine tophospholipids 1 5 Addition of calcium no yes no yes NATHIN 250 (g) 10 1010 10 Serine (g) 0.8 0.8 4.0 4.0 Calcium chloride (g) — 0.04 — 0.19Water (g) 2.6 2.6 5.7 5.7 PLD-Y1 (units) 82.2 82.2 82.2 82.2

TABLE 2 Molar ratio of serine to phospholipids 1 5 Addition of calciumno yes no yes PA (mole %) 13.0 11.8 10.9 11.0 PE (mole %) 4.7 3.3 3. 2.7PS (mole %) 34.5 35.5 44.2 47.4 PC (mole %) 31.0 32.2 25.0 21.4 Otherphospholipids (mole %) 16.9 17.1 16.7 17.5

These results show that good yields can be obtained according to thepresent invention regardless of the presence or absence of calcium.Accordingly, the method of the present invention does not require theaddition of calcium to its reaction system in contrast to most ofconventional transphosphatidylation reactions using an organic solvent.

EXAMPLE 3 Reaction with Phospholipid Containing no Triglyceride asSubstrate

A total of 50 g of NATHIN 250 (available from Central Soya Company,Inc.) was dissolved in 500 mL of a reagent chemical acetone by heatingto 60° C., and the solution was cooled to 15° C. The resultingprecipitate was dissolved in 300 mL of a reagent chemical acetone byheating to 60° C., the solution was then cooled to 15° C. and therebyyielded a precipitate comprising phospholipids alone with notriglyceride. To 6 g of a dried product of the precipitate (hereinafterreferred to “NATHIN 250/acetone precipitate”) was kneaded (homogenized)with 5.8 g of 4.5M L-serine aqueous solution (with or without theaddition of 0.18 M calcium chloride), and the kneaded article was heldat 55° C. The kneaded article was further kneaded (homogenized) with 0.2mL of an enzyme mixture containing 49.3 units of Phospholipase D Y-1(available from Yakult Honsha Co., Ltd.) using a microspatula, and thekneaded article was immediately subjected to a reaction at 55° C. for 17hours to thereby complete a transphos-phatidylation reaction. TABLE 3 PSProduction Reaction Using NATHIN 250/acetone Precipitate as Substrate(Substrate Composition) Molar ratio of serine to phospholipids 5Addition of calcium no yes NATHIN 250/acetone precipitate (g) 6.0 6.0Serine (g) 2.4 2.4 Calcium chloride (g) — 0.1 Water (g) 3.4 3.4 PLD-Y1(units) 49.3 49.3

Analyses of obtained reaction products by silica gel thin-layerchromatography revealed that 41.7% of total phospholipids was convertedinto phosphatidylserines when 0.18 M calcium chloride was added.However, 35.1% of total phospholipids was converted intophosphatidylserines even without the addition of calcium chloride.Homogenized mixtures before the initiation of the reaction weresubjected to microscopic observation under crossed Nicols and were foundto have a “lamellar lyotropic liquid crystal structure substantiallywithout phase separation”. Tables 3 and 4 show compositions ofsubstrates and the results of PS transphatidylation reactions using theacetone precipitate as a substrate, respectively. TABLE 4 PS ProductionReaction Using NATHIN 250/acetone Precipitate as Substrate(Phospholipids Compositions of Purified Products) Molar ratio of serineto phospholipids 5 Addition of calcium no yes PA (mole %) 9.0 10.2 PE(mole %) 2.7 1.9 PS (mole %) 35.1 41.7 PC (mole %) 35.3 28.8 Otherphospholipids (mole %) 17.9 17.4

EXAMPLE 4

Raw material phospholipids were prepared by kneading 1 to 9% by weightof MCT (Panasate 810, trademark, available from Nippon Oil And Fats Co.,Ltd.) into NATHIN 250/acetone precipitate. Each of the materialphospholipids was further kneaded with 200 mg of serine and 200 mL ofwater (molar ratio of phospholipids to water 0.4) relative to 500 mg ofNATHIN 250/acetone precipitate. The kneaded article was further kneaded(homogenized) with a PLD-Y1 aqueous solution (4.1 units per 15 mL) usinga microspatula and was allowed to react at 55° C. for 17 hours.Phosphatidylserines after the completion of reaction were determined bysilica gel thin-layer chromatography.

FIG. 1 is a diagram showing test results on optimum amounts of MCT. Asshown in FIG. 1, by adding 50 mg (9% relative to phospholipids) of MCT,the PS production rate increases from 34.7% to 40.4% as compared withthe case where MCT is not added, verifying effects of the addition ofMCT. Reactions were performed in the same manner with a varying ratio ofMCT of 10% to 40%. FIG. 2 is a diagram showing test results on optimumamounts of MCT. As shown in FIG. 2, the PS production rate increases inall the samples with 10% to 40% of MCT. Apparently. FIGS. 1 and 2indicate that the amount of MCT is preferably from 9% to 40%.Homogenized mixtures before the initiation of the reaction were observedwith a microscope under crossed Nicols and were found to have a“lamellar lyotropic liquid crystal structure substantially without phaseseparation”.

EXAMPLE 5 Molar Ratio of Serine to Phospholipids

To 500 mg of NATHIN 250 was added 2.5 M serine aqueous solutioncontaining 0.18 M calcium chloride in a varying molar ratio of serine tophospholipids from 0.1 to 15, and the mixture was mixed in a mixer whilewarming on a water bath at 60° C. After cooling the water bath to 55°C., the reaction substrate was further mixed and homogenized with aPLD-Y1 aqueous solution using a microspatula or Vibromixer. Thehomogenized mixture was allowed to react at 55° C. for 17 hours, and thephospholipid composition of reaction products was analyzed by silica gelthin-layer chromatography.

FIG. 3 shows that the PS content in the reaction products is 20% or moreat molar ratios of serine to phospholipids of 0.3 or more, indicatingthat these are suitable conditions for the production of PS. Inparticular, molar ratios of 4 or more can achieve PS contents of 45% ormore and are typically preferred as substrate compositions. However, ifthe molar ratio is 10 or more, the production of PS does not increasewith an increasing amount of serine, indicating that an upper limit ofthe molar ratio for efficient production of phosphatidylserines is about10. Homogenized mixtures (at molar ratios from 0.3 to 10) beforereaction were subjected to microscopic observation under crossed Nicolsand were found to have a “lamellar lyotropic liquid crystal structuresubstantially without phase separation”.

The amount of water relative to phospholipids was 100 parts by weight ormore at molar ratios of serine of 6 or more. However, the amount ofwater in the homogenized mixtures as determined by the Karl Fischer'stechnique was 100 parts by weight or less after removal of separatedwater by decantation.

EXAMPLE 6 Weight Ratio of Water to Phospholipids

A total amount of 200 mg each of powdery L-serine was sufficiently mixedwith 500 mg of soybean lecithin (NATHIN 250/acetone precipitate, or PC80 available from Croklaan B. V.) or egg yolk lecithin (PL-100LE,available from Q.P. Corporation) in a mortar heated at 60° C. Themixture was further kneaded with distilled water in water contents shownin FIGS. 4 to 6 and was held at 55° C. A total of 0.015 mL of an enzymemixture containing 4.1 units of PLD-Y1 (available from Yakult HonshaCo., Ltd.) was kneaded (homogenized) with the kneaded article using amicrospatula, and a transphosphatidylation reaction was performed at 55°C. for 17 hours while left stand and was complete. The water contents inthe homogenized mixtures were determined by the Karl Fischer'stechnique.

Obtained reaction products were analyzed by silica gel thin-layerchromatography. FIGS. 4 to 6 are diagrams of results with the ordinateshowing PS contents (%) in the reaction products and the abscissa(logarithmically plotted) showing the ratio (% by weight) of water tosoybean lecithin.

FIG. 4 shows that when NATHIN 250/acetone precipitate was used as a rawmaterial phospholipid, PS was not produced at a weight ratio of water tothe phospholipids in the homogenized mixture of 3%, but 10% or more ofthe phospholipid was converted into PS at weight ratios from 10% to100%, indicating that these weight ratios are suitable for theproduction of PS. A total of 30% or more of the phospholipid wasconverted into PS at weight ratios of water to the phospholipids from20% to 70%, indicating that these weight ratios are optimum for theproduction of PS. Homogenized mixtures were subjected to microscopicobservation under crossed Nicols and were found to have a “lamellarlyotropic liquid crystal structure substantially without phaseseparation” at weight ratios of water to the phospholipids from 10% to100%, suitable for the production of PS.

FIG. 5 shows that when PC 80 was used as a raw material phospholipid, PSwas not produced at a weight ratio of water to the phospholipids in thehomogenized mixture of 8%, but 10% or more of the phospholipid wasconverted into PS at weight ratios from 10% to 60%, indicating thatthese weight ratios are suitable for the production of PS. A total of20% or more of the phospholipid was converted into PS at weight ratiosof water to the phospholipids from 20% to 40%, indicating that theseweight ratios are optimum for the production of PS. Homogenized mixtureswere subjected to microscopic observation under crossed Nicols and werefound to have a “lamellar lyotropic liquid crystal structuresubstantially without phase separation” at weight ratios of water to thephospholipids from 10% to 60%, suitable for the production of PS.

FIG. 6 shows that when the egg yolk lecithin was used as a raw materialphospholipid, PS was not produced at a weight ratio of water to thephospholipids in the homogenized mixture of 8%, but 10% or more of thephospholipid was converted into PS at weight ratios from 15% to 40%,indicating that these weight ratios are suitable for the production ofPS. A total of 20% or more of the phospholipid was converted into PS atweight ratios of water to the phospholipids from 20% to 35%, indicatingthat these weight ratios are optimum for the production of PS.Homogenized mixtures were subjected to microscopic observation undercrossed Nicols and were found to have a “lamellar lyotropic liquidcrystal structure substantially without phase separation” at weightratios of water to the phospholipids from 15% to 40%, suitable for theproduction of PS.

EXAMPLE 7 Other Than PS

Each 490 mg of aqueous solutions of acceptors indicated in Table 5 wasmixed with 500 mg of PC 80 on a water bath at 60° C., the mixture waskneaded (homogenized) with 50 mL of an aqueous solution containing 13.7units of a PLD (PLD-Y1) using a microspatula, and the kneaded articlewas allowed to react at 55° C. for 18 hours. Transphosphatidylationreaction products were analyzed by silica gel thin-layer chromatography.TABLE 5 Transfer Reactions Using Acceptors other than SerineConcentration of Production rate of aqueous solution acceptortransferred Acceptor as used % (w/w) pH lecithin (%) Glycerol 50 5.942.7 L-Ascorbic acid 41 5.0 9.4 Magnesium ascorbate 17 6.7 0.0 phosphateester Sodium ascorbate 50 7.0 0.0 phosphate ester Inositol 17 6.5 0.0Glucose 50 5.0 13.3 Trehalose 50 5.3 0.0

Table 5 shows production rates of transferred products after transferreactions using each of the acceptors. Table 5 demonstrates thatcorresponding transphosphatidylated products (phosphatidylglycerol:42.7%, phosphatidylascorbic acid: 9.4%, phosphatidylglucose: 13.3%) wereproduced when glycerol, L-ascorbic acid, and glucose were each used asthe acceptor. In contrast, no transphosphatidylated product was producedwhen inositol, ascorbate phosphates, and trehalose were used.

EXAMPLE 8 Reaction Temperatures

A total of 490 mg each of 4.5 M serine aqueous solution was added to 500mg of NATHIN 250/acetone precipitate (PL) or of a phospholipid substrate(PL+TG) comprising 450 mg of NATHIN 250/acetone precipitate and 50 mg ofMCT, the resulting mixture was kneaded at 60° C., was further kneaded(homogenized) with 15 mL of an aqueous solution containing 4.1 units ofPLD-Y1 using a microspatula and was allowed to react at temperaturesfrom 15° C. to 65° C. in increments of 10° C. for 18 hours.Transphosphatidylated products were analyzed by silica gel thin-layerchromatography.

FIG. 7 is a diagram showing production rates of PS at different reactiontemperatures and shows that the production rate of PS at 45° C. was 43%and did not change with an elevating temperature when the substratecontaining NATHIN 250/acetone precipitate and MCT was used.

When the substrate containing NATHIN 250/acetone precipitate alone wasused, the production rate of PS was 30.1% at 45° C. and 38.7% at 65° C.,indicating that a more satisfactory result was obtained with anincreasing reaction temperature. However, it is speculated that theupper limit of the reaction temperature is 65° C., since PS productiondecreases and the phospholipids may be deteriorated or decomposed atreaction temperatures exceeding 65° C.

EXAMPLE 9 Brussels Sprout PLD

A total of 490 mg of 4.5 M serine aqueous solution (containing, if any,9.3 mg of calcium) was mixed with 500 mg of NATHIN 250 at 55° C. Aftercooling to 45° C., the reaction mixture was treated with 200 mL of aliquid enzyme mixture (0.6 units per milliliter) of Brussels sprout PLDat 45° C. for 18 hours. The Brussels sprout PLD had been separatelyprepared according to a conventional procedure.

As a result, a reaction did hardly proceed without the addition ofcalcium, but proceeded and thereby yielded reaction products containing6.2% of PS with the addition of calcium.

EXAMPLE 10 Concentration of Phosphatidylserines; No. 1

With 200 g of soybean lecithin containing 40% of PC were kneaded 190 gof a serine aqueous solution containing 70 g of serine in 120 g of waterand 10 mL of an aqueous solution (24 mg/mL) of phospholipase D (PLD-Y1,available from Yakult Honsha Co., Ltd.). The kneaded article was allowedto react at 55° C. for 5 hours and thereby yielded a reaction productcontaining 46.7% of PS in phospholipids.

A total of 5.0 g of the reaction product was extracted with 20 mL ofethyl alcohol at 45° C., and the residue (precipitate) was furtherextracted with two portions of 5 mL of ethyl alcohol. Three extractswere mixed, 5 mL of which was treated with 0.20 mL of 25% common salt(sodium chloride) aqueous solution, was heated to 45° C., was left standat room temperature and thereby yielded a precipitate. As a result, theprecipitate contained 62.1% of PS on dried solid matter basis, whereas asupernatant contained 3.3% of PS, indicating that PS was efficientlyconcentrated in the precipitated.

EXAMPLE 11 Concentration of Phosphatidylserines; No. 2

A total of 50 mg of powdery sodium acetate was added to 5 mL of themixture of the extracts obtained in Example 10, was heated to 45° C.,was left stand at room temperate and thereby yielded a precipitate. As aresult, the precipitate contained 61.8% of PS on dried solid matterbasis, whereas a supernatant contained 3.5% of PS, indicating that PSwas efficiently concentrated in the precipitate as in the use of thecommon salt aqueous solution.

EXAMPLE 12 Concentration of Phosphatidylserines; No. 3

A 25% common salt aqueous solution was added to the mixture of theextracts obtained in Example 10 and thereby yielded insolubilized PSs.The amounts of PSs in the precipitated phospholipids and in therecovered precipitate were determined. Table 6 shows the relationshipamong the amount of common salt, the recovery rate of PS in theprecipitate and the PS content in the phospholipids.

As shown in Table 6, PS was concentrated in a precipitated fractionunder any conditions, and among them, the PS content in the precipitatedphospholipids was 55% or more at amounts of common salt of 10 mmol orless per gram of the phospholipids in the extracts mixture, indicatingthat PS was efficiently concentrated in the precipitate. The recoveryrate of PS into the precipitate was 60% or less, and 40% or more thereofremained in the supernatant at amounts of common salt of 0.05 mmol orless. These results show that PS can be concentrated into a precipitateat any amount of common salt but is practically sufficientlyconcentrated at amounts of common salt from 0.15 to 10 mmol per gram ofphospholipids dissolved in an alcohol. TABLE 6 Relation between Amountsof Common Salt and PS Recovery Rate/PS Contents in Phospholipids CommonPS content in PS content in salt*¹⁾ PS recovery precipitated supernatant(mmol/g) rate*²⁾ (%) PL*³⁾ (%) PL*⁴⁾ (%) None — — 43.2 0.05 53.7 64.733.4 0.15 73.8 66.7 28.2 0.25 80.4 69.3 18.0 0.50 96.2 63.8 6.2 1.2597.4 63.5 4.3 2.5 96.6 62.7 6.1 5 97.1 59.8 6.4 10 95.7 55.0 10.5 2597.8 47.2 12.0 50 97.7 46.6 13.5*¹⁾Ratio of amount of common salt per unit weight of phospholipids(mmol/g)*²⁾PS recovery rate into precipitated fractions (%)*³⁾PS content in precipitated phospholipids (%)*⁴⁾PS content in supernatant phospholipids (%)

1. A method for producing a phospholipid by transphosphatidylationcomprising: homogenizing a mixture of a raw material phospholipid, ahydroxyl-containing acceptor, phospholipase D and water in the absenceof an organic solvent to obtain a homogenized mixture; and subjectingsaid homogenized mixture to a transphosphatidylation reaction at atemperature within a range from 15° to 65° C.
 2. The method according toclaim 1, wherein said homogenized mixture substantially has a lamellarlyotropic liquid crystal structure.
 3. The method according to claim 1,wherein the content of water in said homogenized mixture is adjustedwithin a range from 10 wt % to 100 wt % relative to the weight of theraw material phospholipid.
 4. The method according to claim 1, whereinthe content of said hydroxyl-containing acceptor is adjusted within arange from 0.3 mole to 10 moles per 1 mole of the raw materialphospholipid in said homogenization.
 5. The method according to claim 1,wherein said hydroxyl-containing acceptor is at least onehydroxyl-containing acceptor selected from the group consisting ofserine, glycerol, L-ascorbic acid, glucose and choline.
 6. The methodaccording to claim 1, wherein said hydroxyl-containing acceptor isserine, which yields a phosphatidylserine.
 7. The method according toclaim 1, which further comprises adding an edible oil and/or a fatduring said homogenization.
 8. The method according to claim 6, furthercomprising the steps of: dissolving the phospholipid which contains saidphosphatidylserine in an alcohol to obtain a solution; and carrying outan insolubilization by adding a metallic salt to said solution toinsolubilize the phosphatidylserine and separating the resultantinsolubilized matter.
 9. The method according to claim 8, wherein saidmetallic salt is at least one metallic salt selected from the groupcomprising a lithium salt, a potassium salt, and a sodium salt.
 10. Themethod according to claim 8, wherein said metallic salt is lithiumchloride, potassium chloride, or sodium chloride.
 11. The methodaccording to claim 8, wherein the alcohol is ethyl alcohol.